BIOCHEMISTRY
Effect of Neutral Salts on Enzyme Activity and Structure* James C. Warrent and Sarah G. Cheatum
When trypsin, a-chymotrypsin, renal acylase, wheat germ lipase, estradiol-17P dehydrogenase, pamylase, and &galactosidase utilize uncharged substrates, neutral salts inhibit their activity in an order of increasing effectiveness for anions: CH,COO- < C1< Br- i X Nos- < I- < C10,- SCN-. This order is essentially similar to the order in which these anions disrupt the structure of diverse macromolecules and inhibit the activity of various enzymes utilizing charged substrates. Neutral salts, at concentrations which only partly inhibit the activity of P-amylase, increase the rate ABSTRACT:
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of reaction of its sulfhydryl groups with 5 3 '-dithiobis(2-nitrobenzoic acid) in an order of increasing effectiveness for anions: CHsCOO- < C1- < c104- < SCN-, which is similar to the inhibition order. These salts increase the reaction rate of P-amylase sulfhydryl groups to a degree which exceeds their effects on the reaction rate of the sulfhydryl groups of reduced glutathione for which the order of effectiveness is also different. These observations provide additional evidence that salts alter enzyme activity by altering the organized structure of the protein macromolecule.
t has been recently demonstrated (Warren et af., 1966) that neutral salts at high concentrations inhibit the activity of widely different enzymes in an order of increasing effectiveness for anions: Ac- < C1- < N03< Br- < I- < SCN- < c104- and an order of increasing effectiveness for cations: (CH3)4N+ < Cs+ < K+ < Na+ < Li+. Because the orders are similar to those in which these ions disrupt the structure of diverse macromolecules as determined by physical methods (von Hippel and Wong, 1964), it seems plausible that the observed inhibition may result, at least in part, from disruption of organized enzyme structure. Although the orders of effectiveness for inhibition of activity and disruption of structure of myosin were similar, the ranges were different in that activity was generally completely lost at salt concentrations lower than those at which optical rotatory dispersion indicated a definitive decrease in helix content (Warren et a f . , 1966; Tonomura et a f . ,1962). This investigation was carried out for two reasons. First, to evaluate the anion series on a group of enzymes which uses uncharged substrates to establish the order of effectiveness and allow a general comparison of magnitude of inhibition with the previously studied group which utilized charged substrates. We considered that when the substrate is uncharged, electrostatic shielding effects at the active site would be minimized and effects on activity more clearly related to structural change. Second, to examine the possibility that an indication of conformational change can be
Materiufs. Reagent grade salts, purchased from the Mallinckrodt Co. or G. Frederick Smith Co. (NaC104), were used without further purification. PNPA,' estradiol-17P and its 3-methyl ether, estrone sulfate, Tris base, maleic acid, NADP+, BAPNA, PNPG, GSH, EDTA, and guanidine hydrochloride were from the Sigma Co. The 3,5-nitrosalicylic acid was from Eastman Organic Chemicals Co. Ellman's reagent (DTNB) was obtained from the Aldrich Chemical Co. Distilled, deionized water was used in the preparation of all solutions. Estradiol 3-sulfate was prepared by reduction of estrone 3-sulfate with sodium borohydride (Warren and Salhanick, 1961) and purified by Celite chromatography (Siiteri et al., 1963). After hydrolysis, chromatography in the benzene-formamide system of Zaffaroni (1953) revealed < O S estrone. Enzymes and Assays. Sweet potato P-amylase (two times recrystallized) and trypsin (containing
